Figure 6

Fgf9-induced increase of Nanos2 expression depends on Nodal signaling and is inhibited by Kl. (a) Left panel: Semiquantitative RT-PCR analysis of mRNA expression of Nanos2 in untreated Kit+ spermatogonia and in the same cells cultured overnight with Fgf9, in the presence or absence of the Alk4/7 selective inhibitor SB431542. In this experiment, we used Nanos2 primers '1' listed in Supplementary Table 1. Right panel: Semiquantitative RT-PCR analysis of mRNA expression of Nanos2 in untreated Kit+ spermatogonia and in the same cells cultured overnight with Kl, with Fgf9 or with both growth factors. In this experiment, we used Nanos2 primers ‘2’ listed in Supplementary Table 1. For both panels, the results of densitometric analysis from three independent experiments is shown below. Bars represent the mean ±S.D. (b) Schematic representation which summarizes the main findings of our present work: Fgf9 maintains differentiating (Kit+) spermatogonia in the mitotic condition and prevent meiosis (Stra8 and Scp3 expression) through a cascade involving sustained Erks activation and the consequent stimulation of the Cripto-Nodal-pSmad2/3 signaling, which leads to expression of the meiotic gatekeeper Nanos2. On the opposite, retinoic acid (RA) and Kl/Kit signaling inhibit Nodal expression and stimulate Pi3k-Akt signaling, which is required for Stra8 and Scp3 expression, with the consequent mitotic-meiotic switch